Pathogenicity Detection and Genome Analysis of Two Different Geographic Strains of BmNPV

Simple Summary The hemolynamic septic disease in silkworms is caused by Bombyx mori nuclear polyhedrosis virus (BmNPV). It is the most severe viral disease that adversely affects the sericulture industry. Breeding BmNPV-resistant silkworm varieties is the most economic and effective solution. However, BmNPVs from different geographical strains have different pathogenicities. This brings the challenges of cultivating BmNPV-resistant silkworm varieties with wider adaptabilities. In this study, the genomes of two BmNPV strains (BmNPV ZJ and BmNPV YN) were sequenced and characterized to compare the difference in pathogenicity between the two strains. A total of 76 different genes in these two viruses were found with amino acid mutations. These included genes were associated with BmNPV replication and infection. In addition, the relative gene expression of the BmNPV YN strain was lower than that BmNPV ZJ. Thus, we speculate that the mutations in some genes may affect viral functions and may be the cause of the higher pathogenicity of BmNPV YN despite its lower proliferation rate. The present research provides new clues for further exploring the mechanism determining the difference in pathogenicity of different BmNPV strains. Abstract The pathogenicity of different concentrations of Bombyx mori nuclear polyhedrosis virus- Zhenjiang strain (BmNPV ZJ) and Yunnan strain (BmNPV YN) was assessed in Baiyu larvae. The structures of the two viral strains were observed by negative-staining electron microscopy, and their proliferation was examined by quantitative polymerase chain reaction (qPCR). The genomic sequences of these two viruses were obtained to investigate the differences in their pathogenicity. The lethal concentration 50 (LC50) of BmNPV ZJ against Baiyu larvae was higher than that of BmNPV YN, indicating a relatively more robust pathogenicity in BmNPV YN. Electron microscopic images showed that the edges of BmNPV YN were clearer than those of BmNPV ZJ. The qPCR analysis demonstrated significantly higher relative expressions of immediately early 1 gene (ie-1), p143, vp39, and polyhedrin genes (polh) in BmNPV ZJ than in BmNPV YN at 12–96 h. The complete genomes of BmNPV ZJ and BmNPV YN were, respectively, 135,895 bp and 143,180 bp long, with 141 and 145 coding sequences and 40.93% and 39.71% GC content. Considering the BmNPV ZJ genome as a reference, 893 SNP loci and 132 InDel mutations were observed in the BmNPV YN genome, resulting in 106 differential gene sequences. Among these differential genes, 76 (including 22 hub genes and 35 non-hub genes) possessed amino acid mutations. Thirty genes may have been related to viral genome replication and transcription and five genes may have been associated with the viral oral infection. These results can help in understanding the mechanisms of pathogenicity of different strains of BmNPV in silkworms.


Introduction
Bombyx mori nuclear polyhedrosis virus (BmNPV), a circular double-stranded DNA virus, is the first insect baculovirus to be discovered [1,2]. The B. mori hemolynamic septic disease caused by BmNPV is the most severe viral disease in silkworms that harms the sericulture industry [3]. It is a subacute disease that occurs over 3-5 days, and the onset is faster in summer and autumn. The typical characteristics of infected silkworms include manic creeping, the body becoming whitish and shiny with intersegmental swelling, the skin easily breaking and oozing a milky white body fluid, and, finally, death. The milky white body fluid contains the BmNPV virus particles, which contaminate the mulberry leaves on which the silkworms reside. The contaminated leaf is eaten by other silkworms, which contract oral infections or infections through the wound after coming into contact with other wounded silkworms. The outbreak of the disease in rural production areas usually occurs in the middle and late stages of the fifth instar of B. mori. It is difficult to prevent and leads to a significant reduction in the quantity of production and, sometimes, no harvest occurs. Cultivating silkworm varieties with high resistance to BmNPV is the most economical and effective means to reduce the loss in sericulture production. Over the last 10 years, various institutions in China have cultivated several silkworm varieties resistant to BmNPV [4][5][6][7]; for example, "Huakang No.2", which exhibits more than 100 times improved resistance to B. mori hemolynamic septic disease of the typical summer and autumn species "Qiufeng × Baiyu". The silk productivity of these varieties also indicates their remarkable disease resistance properties [8][9][10][11].
Like in other viruses, genetic variation has also been found in BmNPV [12][13][14] and characterizes the different geographical strains [15,16]. In addition, different geographic strains of BmNPV have differences in their morphology and pathogenicity [17,18]. Even the virulence of local strains of BmNPV from the same province also differs against B. mori. Bai et al. [19] and Tang et al. [12] found that the pathogenicity of eight strains of BmNPV in samples from various regions in Yunnan differed and exhibited varying rates of infectivity in B. mori. Wang et al. [20] reported that the pathogenicity of BmNPV from different origins in Guangxi Province was also distinct in different silkworm varieties, which brought a new challenge to the breeding of silkworms against BmNPV. It is therefore necessary to explore the pathogenic mechanisms of different mutant strains of BmNPV in relation to silkworms.
We isolated two BmNPVs from Zhenjiang, Jiangsu Province, and Luliang, Yunnan Province, with varying pathogenicities to the same silkworm varieties. In the present study, we applied Illumina second-generation sequencing technology and Pacbio thirdgeneration sequencing technology to characterize their genomes and explore the underlying mechanism of the variation in pathogenicity of different BmNPV strains.

Materials
BmNPV strains were obtained from different geographic regions: the BmNPV Zhenjiang strain (BmNPV ZJ) was obtained from Zhenjiang, Jiangsu province, and the BmNPV Yunnan strain (BmNPV YN) was obtained from Luliang, Yunnan province. These two strains were isolated, purified, and preserved by our teams.
The silkworm variety "Baiyu", bred by the Institute of Sericulture of the Chinese Academy of Agricultural Sciences, was the parent of the control variety of silkworm varieties approved by the state for use in summer and autumn and was maintained in our laboratory.

Virus Collection and Purification
The budded virus (BV) of BmNPV ZJ was preserved and provided by the Pathology Laboratory of the Institute of Sericulture of the Chinese Academy of Agricultural Sciences. The BV of BmNPV YN was preserved and provided by the Institute of Sericulture of the Yunnan Academy of Agricultural Sciences. In the autumn of 2019, our team used the BV to puncture the fifth instar of silkworms. After the onset of the disease, the blood of the infected silkworm was collected, filtered, and centrifuged three to four times to obtain the purified virus solution. Then, it was diluted with an appropriate amount of double-distilled water and counted using a hemocytometer. The concentrations of BmNPV ZJ and BmNPV YN were 2.45 × 10 9 and 3.66 × 10 9 , respectively; they were stored at 4 • C for further use.

Determination of Pathogenicity of Different BmNPV to B. mori
The original viral solutions of the two BmNPV strains were diluted with sterile water to a total of five concentrations, from 1 × 10 4 -1 × 10 8 , and smeared on mulberry leaves to feed the Baiyu larvae. The mulberry leaves with smooth surfaces were cut into small pieces (2.7 cm × 2.7 cm) using a punch, and 50 µL of different concentrations of polyhedral suspension was added dropwise on the upper surface of each leaf piece. After smearing evenly, these were fed to the second instar larvae of the silkworm, and leaves with the same volume of sterile water were used as blank controls. There were a total of 90 silkworm larvae per treatment, with three replicates (30 silkworms each), for each BmNPV concentration [20]. Four mulberry leaves carrying the virus were fed to each silkworm in each area at one time. After 24 h, the mulberry leaves carrying the virus were replaced with plain mulberry leaves with no virus. The growth and development of silkworms were observed every day, and the diseased silkworms were removed in time to avoid cross-infection. The number of dead silkworms that were infected with BmNPV was recorded after their third instar stage. The lethal concentration 50 (LC 50 ) of Baiyu was calculated using the Statistical Product and Service Solutions (SPSS, https://www.ibm.com/cn-zh/analytics/spss-statistics-software accessed on 26 September 2021), and the pathogenicities of BmNPV YN and BmNPV ZJ were compared.

Negative-Staining Electron Microscopy to Observe BmNPV Particles
The purified BmNPV ZJ and BmNPV YN were suspended on a copper mesh supported by a polyvinyl alcohol formaldehyde membrane (formvar membrane). The samples were stained with 2% phosphotungstic acid (pH 7.2) for 20 s. After washing and drying, the morphologies of the virus particles were observed with negative-staining electron microscopy.

Sequencing of BmNPV ZJ and BmNPV YN Genomes
DNA was extracted from BmNPV YN and BmNPV ZJ according to the alkaline lysis method [12]. The concentrations of the extracted viral DNA were measured using the Qubit quantitative detector. The DNA quality was assessed using 1% agarose gel electrophoresis. The high-quality viral DNA was used for genome sequencing using Illumina secondgeneration and PacBio third-generation sequencing technologies (Sangon Biotech Co., Ltd., Shanghai, China).

Analysis of BmNPV ZJ and BmNPV YN Genome Sequences
Using the genome of BmNPV T3 (L33180.1, 128,413 bp) as the reference, the sequencing data were assembled and corrected by SPAdes (https://cab.spbu.ru/software/spades/ accessed on 26 September 2021) and Primer-initiated Sequence Synthesis for Genomes (PrlnSeS-G, https://updeplasrv1.epfl.ch/prinses/ accessed on 26 September 2021) [21,22]. The Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups (COGs) of proteins, non-redundant proteins (NR), curated protein families (PFAM), Swiss-Prot, and TrEMBL databases were used for functional annotation of the virus genes. The genome sequences of these two BmNPVs were compared with other published Bm-NPV genome sequences. For multiple sequence alignment, the ClustalW parameters of Molecular Evolutionary Genetics Analysis (MEGA 7.1, https://megasoftware.net/ accessed on 26 September 2021) were used. The baculovirus repeated orfs gene (bro) of the BmNPV exists in diverse copies and nucleotide sequences in different ecological environments; therefore, this gene was also used for molecular identification of different strains of BmNPV [15,16,23]. The bootstrap statistical method was used for the calculation of

Detection of Gene-Relative Expression of BmNPV by qPCR
The two strains of BmNPV were diluted to 1 × 10 8 and fed to the fifth instar of the Baiyu silkworms, feeding 7 µL to each silkworm; there were five silkworms per treatment. The disease incidence rate among the silkworms was 100% at this concentration. At 12,24,48,72, and 96 h, the silkworms were dissected and RNA was extracted from their midgut tissues. The very early gene ie-1, early gene p143, late gene vp39, and very late gene polh of BmNPV were chosen for quantitative polymerase chain reaction (qPCR) analysis. The primers for viral genes were designed in Primer 6.0 and their details are listed in Table 1. Reaction conditions for qPCR were: 95 • C, 30 s; 95 • C, 10 s; and 55 • C, 30 s, for a total of 40 cycles. Using actin-3 (U49854) as the reference gene, the relative expression levels of the genes in the two BmNPV strains were calculated with the 2 −∆∆CT method.  Referring to the design process proposed by the Genome Analysis Toolkit (GATK) [24], the effective data for the BmNPV YN and BmNPV ZJ genomes were compared using the Burrows-Wheeler Aligner (BWA, http://bio-bwa.sourceforge.net/ accessed on 26 September 2021). To convert and sort the results, sequence alignment/map Tools (SAMtools, http://www.htslib.org/doc/samtools.html accessed on 26 September 2021) was used and the comparison results were statistically analyzed. The genotype differences between Bm-NPV YN and BmNPV ZJ were assessed using the HaplotypeCaller software, and the single nucleotide polymorphism (SNP) and insertion and deletion (InDel) information of the two strains were obtained. After quality control (Table 2), the SNP and InDel information were annotated using SNP effect software (SnpEff, https://pcingola.github.io/SnpEff/ accessed on 26 September 2021) [25]. According to the annotation results and BLAST analysis of the differential genes between BmNPV YN and BmNPV ZJ, candidate genes associated with the difference in pathogenicity of the BmNPV YN and BmNPV ZJ were screened.

The Pathogenicity of BmNPV ZJ and BmNPV YN to B. mori
The mortality rate of Baiyu larvae increased with the increase in the concentration of BmNPV ZJ and BmNPV YN (Figure 1). At the same concentration, BmNPV YN exhibited stronger pathogenicity than BmNPV ZJ. In addition, the mortality rates at 1 × 10 7 and 1 × 10 8 of both BmNPVs differed significantly.

The Pathogenicity of BmNPV ZJ and BmNPV YN to B. mori
The mortality rate of Baiyu larvae increased with the increase in the concentrat BmNPV ZJ and BmNPV YN (Figure 1). At the same concentration, BmNPV YN exh stronger pathogenicity than BmNPV ZJ. In addition, the mortality rates at 1 × 10 7 × 10 8 of both BmNPVs differed significantly.
The median lethal dose of the two viruses in the Baiyu larvae was calculated by v21 (https://www.ibm.com/cn-zh/analytics/spss-statistics-software accessed on 26 tember 2021) software. The LC50 concentration of BmNPV YN in Baiyu larvae was 10 times less than that of BmNPV ZJ (Table 3), indicating that BmNPV YN could cause death in half of these larvae at a low concentration.

The Morphology and Size of BmNPV ZJ and BmNPV YN
The purified BmNPV ZJ and BmNPV YN virus particle suspensions were obs by electron microscopy ( Figure 2). The sizes of BmNPV ZJ and BmNPV YN appeare ilar, with diameters of about 2.2-4.0 μm. The BmNPV polyhedra appeared mostly h onal, while a small number of particles appeared quadrilateral and irregular. The of BmNPV YN particles appeared sharper compared with those of BmNPV ZJ. The median lethal dose of the two viruses in the Baiyu larvae was calculated by SPSS v21 (https://www.ibm.com/cn-zh/analytics/spss-statistics-software accessed on 26 September 2021) software. The LC 50 concentration of BmNPV YN in Baiyu larvae was about 10 times less than that of BmNPV ZJ (Table 3), indicating that BmNPV YN could even cause death in half of these larvae at a low concentration.

The Morphology and Size of BmNPV ZJ and BmNPV YN
The purified BmNPV ZJ and BmNPV YN virus particle suspensions were observed by electron microscopy (Figure 2). The sizes of BmNPV ZJ and BmNPV YN appeared similar, with diameters of about 2.2-4.0 µm. The BmNPV polyhedra appeared mostly hexagonal, while a small number of particles appeared quadrilateral and irregular. The edges of BmNPV YN particles appeared sharper compared with those of BmNPV ZJ.

The Relative Expression of Genes of BmNPV ZJ and BmNPV YN in the Midgut of Baiyu
The qPCR analysis of the genes of BmNPV ZJ and BmNPV YN in the midgut of Baiyu larvae ( Figure 3A) revealed that the relative expression of ie-1 in BmNPV ZJ was higher than that in BmNPV YN at 12-96 h, with the highest level at 96 h. The expressions of p143, vp39, and polh in BmNPV ZJ were also higher than BmNPV YN before 96 h ( Figure 3B-D).

The Relative Expression of Genes of BmNPV ZJ and BmNPV YN in the Midgut of Baiyu
The qPCR analysis of the genes of BmNPV ZJ and BmNPV YN in the midgut of Baiyu larvae ( Figure 3A) revealed that the relative expression of ie-1 in BmNPV ZJ was higher than that in BmNPV YN at 12-96 h, with the highest level at 96 h. The expressions of p143, vp39, and polh in BmNPV ZJ were also higher than BmNPV YN before 96 h ( Figure 3B-D). (D) polh. Note: "*" represents p < 0.05; "**" represents p < 0.01.

Genome Characteristics of BmNPV ZJ
The genome of BmNPV ZJ was estimated to be 135,895 bp long, with 40.39% GC content. It comprised 141 predicted protein-coding sequences (CDSs), accounting for 83.86% of the entire genome sequence, of which 70 CDSs were on the positive strand and 71 on the negative strand. The average length of this genome was estimated to be 808 bp. Further analysis indicated 91 putative genes of 500 to 1000 bp lengths and 40 genes with ≥1000 bp lengths. Only 10 genes were less than 500 bp in length. In total, 139 genes were annotated. The annotated genes fibroblast growth factor (fgf), chi-tinase gene (chi-a), and alkaline exonuclease gene (alk-exo) were found in the BmNPV ZJ genome, but their homologous reading frames were absent in BmNPV YN. Further, late expression factor 12 (lef-12) and AcMNPV 94K were not annotated in the BmNPV T3 reference strain ( Figure S1). Compared with the genome of BmNPV T3, some genes in these two viral genomes had

Genome Characteristics of BmNPV ZJ
The genome of BmNPV ZJ was estimated to be 135,895 bp long, with 40.39% GC content. It comprised 141 predicted protein-coding sequences (CDSs), accounting for 83.86% of the entire genome sequence, of which 70 CDSs were on the positive strand and 71 on the negative strand. The average length of this genome was estimated to be 808 bp. Further analysis indicated 91 putative genes of 500 to 1000 bp lengths and 40 genes with ≥1000 bp lengths. Only 10 genes were less than 500 bp in length. In total, 139 genes were annotated. The annotated genes fibroblast growth factor (fgf ), chi-tinase gene (chi-a), and alkaline exonuclease gene (alk-exo) were found in the BmNPV ZJ genome, but their homologous reading frames were absent in BmNPV YN. Further, late expression factor 12 (lef-12) and AcMNPV 94K were not annotated in the BmNPV T3 reference strain ( Figure S1). Compared with the genome of BmNPV T3, some genes in these two viral genomes had SNP mutations, resulting in premature stop codons, and truncated or abnormal proteins. One putative gene with an unknown function was also found.

Genome Characteristics of BmNPV YN
The genome of BmNPV YN was estimated to be 143,180 bp long, with 39.71% GC content. It comprised 146 CDSs, accounting for 89.37% of the whole genome sequence, of which 76 CDSs were on the positive strand and 69 on the negative strand. The average length of this genome was estimated to be 882.52 bp, with 96 genes of 500 to 1000 bp lengths and 43 genes more than 1000 bp in length. Six genes were found to be of less than 500 bp in length and 137 genes were annotated. Further analysis revealed Orf20, late expression factor 7 (lef-7), and p26 genes in the BmNPV YN genome, but the homologous genes were absent in the BmNPV ZJ genome. Lef-12 was not annotated in the BmNPV T3 reference strain ( Figure S2). In addition, seven putative genes with unknown functions were found.

Genome Comparison of BmNPV
The genome sequences of the two BmNPVs were compared with that of the BmNPV T3 strain as a reference, as shown in Table 4. The nucleotide sequences of the BmNPV ZJ and BmNPV YN genomes were analyzed by DNAMAN software (https://www.lynnon.com/dnaman.html accessed on 26 September 2021). The genes of BmNPV ZJ and BmNPV YN were 92.6-100.0% homologous. A comparison with the genome sequences of the other six BmNPV reference strains showed homologies of BmNPV ZJ and BmNPV YN in the range of 92.0-100.0% and 93.3-100.0%, respectively (Table S1). The bro-d gene sequence of BmNPV ZJ and BmNPV YN showed 95.9% homology, and the homologies with the other BmNPV reference strains were 92.1-99.6% and 93.3-97.3%, respectively ( Table 5). The amino acid sequence homology of the BRO-D (encoded by the bro-d) of BmNPV ZJ and BmNPV YN was 96.6%, and the homologies with other BmNPVs were 92.0-99.7% and 92.2-97.1%, respectively ( Table 5). The amino acid of the BRO-D protein of the two strains was mainly mutated at the N-terminal (Figure 4), which was consistent with the observation of Zhou et al. [26].  The bro-d gene was chosen for the evolutionary analysis of the two BmNPVs. Using Plutella xylostella multiple nucleopolyhedrovirus (PxMNPV) as the exogenous reference, the phylogenetic tree was plotted using the MEGA 7.1 software (https://megasoftware.net/ accessed on 26 September 2021). The results are shown in Figure 5. The eight BmNPV strains were divided into two clades. Both BmNPV ZJ (Zhenjiang) and BmNPV YN (Yunnan) were grouped in clade I, and BmNPV ZJ was closer to the Cubic strain (IQ991009) while BmNPV YN was closer to the India strain (JQ991010). These finding are similar to those reported by Tang et al. [12].

Detection and Analysis of Single Nucleotide Polymorphism and InDels in the Two Viral Genomes
Using BmNPV ZJ as the reference genome, a total of 893 SNP loci and 132 InDel variants were identified in BmNPV YN. The number of SNPs of transitions and transversions were 693 and 195, respectively. The SNP loci of the BmNPV YN strain were mainly enriched in the coding region, accounting for about 82.4% of the total number of SNPs. The bro-d gene was chosen for the evolutionary analysis of the two BmNPVs. Using Plutella xylostella multiple nucleopolyhedrovirus (PxMNPV) as the exogenous reference, the phylogenetic tree was plotted using the MEGA 7.1 software (https://megasoftware.net/ accessed on 26 September 2021). The results are shown in Figure 5. The eight BmNPV strains were divided into two clades. Both BmNPV ZJ (Zhenjiang) and BmNPV YN (Yunnan) were grouped in clade I, and BmNPV ZJ was closer to the Cubic strain (IQ991009) while BmNPV YN was closer to the India strain (JQ991010). These finding are similar to those reported by Tang et al. [12].  The bro-d gene was chosen for the evolutionary analysis of the two BmNPVs. Using Plutella xylostella multiple nucleopolyhedrovirus (PxMNPV) as the exogenous reference, the phylogenetic tree was plotted using the MEGA 7.1 software (https://megasoftware.net/ accessed on 26 September 2021). The results are shown in Figure 5. The eight BmNPV strains were divided into two clades. Both BmNPV ZJ (Zhenjiang) and BmNPV YN (Yunnan) were grouped in clade I, and BmNPV ZJ was closer to the Cubic strain (IQ991009) while BmNPV YN was closer to the India strain (JQ991010). These finding are similar to those reported by Tang et al. [12].

Detection and Analysis of Single Nucleotide Polymorphism and InDels in the Two Viral Genomes
Using BmNPV ZJ as the reference genome, a total of 893 SNP loci and 132 InDel variants were identified in BmNPV YN. The number of SNPs of transitions and transversions were 693 and 195, respectively. The SNP loci of the BmNPV YN strain were mainly enriched in the coding region, accounting for about 82.4% of the total number of SNPs.

Detection and Analysis of Single Nucleotide Polymorphism and InDels in the Two Viral Genomes
Using BmNPV ZJ as the reference genome, a total of 893 SNP loci and 132 InDel variants were identified in BmNPV YN. The number of SNPs of transitions and transversions were 693 and 195, respectively. The SNP loci of the BmNPV YN strain were mainly enriched in the coding region, accounting for about 82.4% of the total number of SNPs. Among them, there were 487 synonymous mutations, 247 missense mutations, and 2 nonsense mutations, resulting in 102 different gene sequences (Table 6). Analysis of InDel mutations by SnpEff software showed that the numbers of InDel mutations were similar between coding and non-coding regions ( Table 7). Further analysis revealed 38 in-frame mutations and 25 frameshift mutations, resulting in 30 differential gene sequences.

Differential Genes Analysis
The BmNPV ZJ and BmNPV YN strains were observed to have 106 different annotated genes sequences, which included non-synonymous mutations, synonymous mutations, and frameshift mutations. Among these, 76 differential nucleotide sequences led to differences in amino acid sequences in the two viruses ( Table 8). The core genes [27] (shared genes of the baculovirus) accounted for 28.94% and non-core genes accounted for 47.37% of all differential genes, while some genes had not been reported earlier. Thirty annotated genes were associated with the viral genome replication and transcription, and five genes were related to the oral infection of viruses. These differences in genes may have been the cause of the difference in pathogenicity between the two virus strains.  The formation of auxiliary components of polyhedra is involved in the transport of virus particles in the cell

Discussion
BmNPV is a serious threat to silkworms and causes huge economic losses to the sericulture industry. Cultivating silkworm varieties resistant to a hemolymph-type septic diseases is the most effective and economical measure to minimize and mitigate these losses [28]. The pathogenicity of BmNPV varies in different geographic regions [18,19]. Furthermore, the underlying molecular mechanism that causes the variations in the pathogenicity of different strains of BmNPV remains unknown and poses a great challenge to the effective prevention of BmNPV. We compared the genomes of two BmNPV strains with different pathogenicities and found differences in some genes related to viral replication and infection.
In this study, the pathogenicities of BmNPV ZJ and BmNPV YN was determined and compared. The LC 50 of BmNPV YN and BmNPV ZJ against Baiyu larvae were 3.62 × 10 5 and 6.45 × 10 6 , respectively, indicating the semi-lethality of BmNPV YN in silkworms at a low concentration. Subsequently, the electron microscopic observation morphologies of virus particles revealed their hexagonal shapes with similar sizes (about 2.2-4.0 µm). These were typical silkworm nuclear polyhedrosis viruses, but the edges of BmNPV YN were clearer than those of BmNPV ZJ. The relative expressions of the ie-1, p143, vp39, and polh genes, expressed, respectively, at the very early, early, late, and very late stages of BmNPV ZJ, in the midgut of Baiyu larvae were higher than those of BmNPV YN within 12-96 h. This further indicates that the lethal concentration of BmNPV ZJ was higher than that of BmNPV YN. After a host is infected by a virus, in general, viruses with a high rate of proliferation tend to exhibit more robust pathogenicity. However, some more lethal virus strains may kill the host even with a lower rate of proliferation [29][30][31]. BmNPV YN might be such a type of lethal strain which, even in lower numbers, can kill the host. This inference is consistent with the finding of a tenfold lower LC 50 for BmNPV YN than for BmNPV ZJ in the second instar of the Baiyu silkworm larvae. Therefore, when the virus was fed at a high concentration (10 8 ) to silkworms, the expression of the virus gene of BmNPV YN was lower, but it was enough to kill the host. Likewise, Ma [32] found that the mutation and recombination in genomes of different strains of Helicoverpa armigera nucleopolyhedrovirus might be the cause of the difference in their pathogenicity even when they are distributed in the same geographic location. Hence, the difference in pathogenicity of our strains might have been because of the variation in their genomes due to the differences in the climates of Zhenjiang, Jiangsu Province, and Luliang, Yunnan Province, which are located, respectively, along the southeast coast and the inland southwest of China.
To further explore the reasons for the difference in pathogenicity between BmNPV YN and BmNPV ZJ to B. mori, the whole genomes of the virus strains were sequenced and analyzed. The genome size of BmNPV ZJ was estimated to be 135,895 bp, with 40.39% GC content and encoding 141 genes, while the genome size of BmNPV YN was estimated to be 143,180 bp, with 39.71% GC content and encoding 145 genes. These data demonstrated that the genome sizes of the two viruses and the numbers of putative genes changed. For further analysis, we used the bro-d gene, which is highly conserved in different strains of BmNPV from varying geographical regions. BmNPV ZJ exhibited the highest homology with the Cubic strains published earlier in the GenBank, which might be due to the closer origin region of BmNPV. These findings suggest that BmNPV from different regions must be used for screening the BmNPV-resistant silkworm varieties for their wider adaptability.
A comparative analysis of BmNPV YN and BmNPV ZJ genomic sequences revealed a large number of SNP and InDel mutations in the two genomes. The nucleotide sequence homology of the two virus genomes was 92.6-100.0%, and a further BLAST analysis revealed 76 different genes between BmNPV YN and BmNPV ZJ. These genes included 22 core genes and 36 non-core genes, mainly the oral infection factor genes, viral replication genes, host molting, and pupation genes. The alterations of genes sequences affect the function of the encoded proteins. Therefore, we speculated that the difference in the genomes of the two viruses might have been the cause of the difference in their pathogenicity in silkworms.
Thus, the roles of viral genes and the mechanism of action of important proteins encoded by these genes need to be studied in the future.

Conclusions
To summarize, the BmNPV YN strain has a relatively lower rate of proliferation but stronger pathogenicity than BmNPV ZJ. The two viruses differ in terms of genome size and the number of coding genes. Among these, 76 genes are different, including genes associated with BmNPV virus replication and infection. These data illustrate that, due to different geographical environments, the genomes of different BmNPV strains mutate and rearrange, which finally leads to differential pathogenicity. The results of this study can provide references to further explore the molecular mechanism relating to the difference in pathogenicity among the viral strains and also provide some guidance in developing new insecticides.